Production of hydroxylated fatty acids in genetically modified plants

ABSTRACT

This invention relates to plant fatty acyl hydroxylases. Methods to use conserved amino acid or nucleotide sequences to obtain plant fatty acyl hydroxylases are described. Also described is the use of cDNA clones encoding a plant hydroxylase to produce a family of hydroxylated fatty acids in transgenic plants. In addition, the use of genes encoding fatty acid hydroxylases or desaturases to alter the level of lipid fatty acid unsaturation in transgenic plants is described.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. patent applicationSer. No. 08/530,862, filed Sep. 20, 1995, the entire contents of whichare hereby incorporated by reference and relied upon.

GOVERNMENT RIGHTS

The invention described herein was made in the course of work undergrant number DE-FG02-94ER20133 from the U.S. Department of Energy andgrant No. MCB9305269 from the National Science Foundation. Therefore,the U.S. Government has certain rights under this invention.

TECHNICAL FIELD

The present invention concerns the identification of nucleic acidsequences and constructs, and methods related thereto, and the use ofthese sequences and constructs to produce genetically modified plantsfor the purpose of altering the fatty acid composition of plant oils,waxes and related compounds.

DEFINITIONS

The subject of this invention is a class of enzymes that introduce ahydroxyl group into several different fatty acids resulting in theproduction of several different kinds of hydroxylated fatty acids. Inparticular, these enzymes catalyze hydroxylation of oleic acid to12-hydroxy oleic acid and icosenoic acid to 14-hydroxy icosenoic acid.Other fatty acids such as palmitoleic and erucic acids may also besubstrates. Since it is not possible to refer to the enzyme by referenceto a unique substrate or product, we refer to the enzyme throughout askappa hydroxylase to indicate that the enzyme introduces the hydroxylthree carbons distal (i.e., away from the carboxyl carbon of the acylchain) from a double bond located near the center of the acyl chain.

The following fatty acids are also the subject of this invention:ricinoleic acid, 12-hydroxyoctadec-cis-9-enoic acid (12OH-18:1^(cisΔ9));lesquerolic acid, 14-hydroxy-cis-11-icosenoic acid (14OH-20:1^(cisΔ11));densipolic acid, 12-hydroxyoctadec-cis-9,15-dienoic acid(12OH-18:2^(cisΔ9,15)); auricolic acid,14-hydroxy-cis-11,17-icosadienoic acid (14OH-20:2^(cisΔ11,17));hydroxyerucic, 16-hydroxydocos-cis-13-enoic acid (16OH22:1^(cisΔ13));hydroxypalmitoleic, 12-hydroxyhexadec-cis-9-enoic (12OH-16:1^(cisΔ9));icosenoic acid (20:1^(cisΔ11)). It will be noted that icosenoic acid isspelled eicosenoic acid in some countries.

BACKGROUND

Extensive surveys of the fatty acid composition of seed oils fromdifferent species of higher plants have resulted in the identificationof at least 33 structurally distinct monohydroxylated plant fatty acids,and 12 different polyhydroxylated fatty acids that are accumulated byone or more plant species (reviewed by van de Loo et al. 1993).Ricinoleic acid, the principal constituent of the seed oil from thecastor plant Ricinus communis (L.), is of commercial importance. We havepreviously described the cloning of a gene from this species thatencodes a fatty acid hydroxylase, and the use of this gene to producericinoleic acid in transgenic plants of other species (see U.S. patentapplication Ser. No. 08/320,982, filed Oct. 11, 1994). The scientificevidence supporting the claims in that patent application weresubsequently published (van de Loo et al., 1995).

The use of the castor hydroxylase gene to also produce otherhydroxylated fatty acids such as lesquerolic acid, densipolic acid,hydroxypalmitoleic, hydroxyerucic and auricolic acid in transgenicplants is the subject of this invention. In addition, the identificationof a gene encoding a homologous hydroxylase from Lesquerella fendleri,and the use of this gene to produce these hydroxylated fatty acids intransgenic plants is the subject of this invention.

Castor is a minor oilseed crop. Approximately 50% of the seed weight isoil (triacylglycerol) in which 85-90% of total fatty acids are thehydroxylated fatty acid, ricinoleic acid. Oil pressed or extracted fromcastor seeds has many industrial uses based upon the properties endowedby the hydroxylated fatty acid. The most important uses are productionof paints and varnishes, nylon-type synthetic polymers, resins,lubricants, and cosmetics (Atsmon 1989).

In addition to oil, the castor seed contains the extremely toxic proteinricin, allergenic proteins, and the alkaloid ricinine. Theseconstituents preclude the use of the untreated seed meal (following oilextraction) as a livestock feed, normally an important economic aspectof oilseed utilization. Furthermore, with the variable nature of castorplants and a lack of investment in breeding, castor has few favorableagronomic characteristics.

For a combination of these reasons, castor is no longer grown in theUnited States and the development of an alternative domestic source ofhydroxylated fatty acids would be attractive. The production ofricinoleic acid, the important constituent of castor oil, in anestablished oilseed crop through genetic engineering would be aparticularly effective means of creating a domestic source.

Because there is no practical source of lesquerolic, densipolic andauricolic acids from plants that are adapted to modern agriculturalpractices, there is currently no large-scale use of these fatty acids byindustry. However, the fatty acids would have uses similar to those ofricinoleic acid if they could be produced in large quantities atcomparable cost to other plant-derived fatty acids (Smith 1985).

Plant species, such as certain species in the genus Lesquerella, thataccumulate a high proportion of these fatty acids, have not beendomesticated and are not currently considered a practical source offatty acids (Hirsinger, 1989). This invention represents a useful steptoward the eventual production of these and other hydroxylated fattyacids in transgenic plants of agricultural importance.

The taxonomic relationships between plants having similar or identicalkinds of unusual fatty acids have been examined (van de Loo et al.,1993). In some cases, particular fatty acids occur mostly or solely inrelated taxa. In other cases there does not appear to be a direct linkbetween taxonomic relationships and the occurrence of unusual fattyacids. In this respect, ricinoleic acid has now been identified in 12genera from 10 families (reviewed in van de Loo et al., 1993). Thus, itappears that the ability to synthesize hydroxylated fatty acids hasevolved several times independently during the radiation of theangiosperms. This suggested to us that the enzymes which introducehydroxyl groups into fatty acids arose by minor modifications of arelated enzyme.

Indeed, as shown herein, the sequence similarity between Δ12 fatty aciddesaturases and the kappa hydroxylase from castor is so high that it isnot possible to unambiguously determine whether a particular enzyme is adesaturase or a hydroxylase on the basis of evidence in the scientificliterature. Similarly, a patent application (PCT/US93/09987) thatpurports to teach the isolation and use of Δ12 fatty acid desaturasesdoes not teach how to distinguish a hydroxylase from a desaturase. Inview of the importance of being able to distinguish between theseactivities for the purpose of genetic engineering of plant oils, theutility of that application is limited to the several instances wheredirect experimental evidence (e.g., altered fatty acid composition intransgenic plants) was presented to support the assignment of function.A method for distinguishing between fatty acid desaturases and fattyacid hydroxylases on the basis of amino acid sequence of the enzyme isalso a subject of this invention.

A feature of hydroxylated or other unusual fatty acids is that they aregenerally confined to seed triacylglycerols, being largely excluded fromthe polar lipids by unknown mechanisms (Battey and Ohlrogge 1989; Prasadet al., 1987). This is particularly intriguing since diacylglycerol is aprecursor of both triacylglycerol and polar lipid. With castormicrosomes, there is some evidence that the pool ofricinoleoyl-containing polar lipid is minimized by a preference ofdiacylglycerol acyltransferase for ricinoleate-containingdiacylglycerols (Bafor et al. 1991). Analyses of vegetative tissues havegenerated few reports of unusual fatty acids, other than those occurringin the cuticle. The cuticle contains various hydroxylated fatty acidswhich are interesterified to produce a high molecular weight polyesterwhich serves a structural role. A small number of other exceptions existin which unusual fatty acids are found in tissues other than the seed.

The biosynthesis of ricinoleic acid from oleic acid in the developingendosperm of castor (Ricinus communis) has been studied by a variety ofmethods. Morris (1967) established in double-labeling studies thathydroxylation occurs directly by hydroxyl substitution rather than viaan unsaturated-, keto- or epoxy-intermediate. Hydroxylation usingoleoyl-CoA as precursor can be demonstrated in crude preparations ormicrosomes, but activity in microsomes is unstable and variable, andisolation of the microsomes involved a considerable, or sometimescomplete loss of activity (Galliard and Stumpf, 1966; Moreau and Stumpf,1981). Oleic acid can replace oleoyl-CoA as a precursor, but only in thepresence of CoA, Mg²⁺ and ATP (Galliard and Stumpf, 1966) indicatingthat activation to the acyl-CoA is necessary. However, no radioactivitycould be detected in ricinoleoyl-CoA (Moreau and Stumpf, 1981). Theseand more recent observations (Bafor et al., 1991) have been interpretedas evidence that the substrate for the castor oleate hydroxylase isoleic acid esterified to phosphatidyicholine or another phospholipid.

The hydroxylase is sensitive to cyanide and azide, and dialysis againstmetal chelators reduces activity, which could be restored by addition ofFeSO₄, suggesting iron involvement in enzyme activity (Galliard andStumpf, 1966). Ricinoleic acid synthesis requires molecular oxygen(Galliard and Stumpf, 1966; Moreau and Stumpf 1981) and requires NAD(P)Hto reduce cytochrome b5 which is thought to be the intermediate electrondonor for the hydroxylase reaction (Smith et al., 1992). Carbon monoxidedoes not inhibit hydroxylation, indicating that a cytochrome P450 is notinvolved (Galliard and Stumpf, 1966; Moreau and Stumpf 1981). Data froma study of the substrate specificity of the hydroxylase show that allsubstrate parameters (i.e., chain length and double bond position withrespect to both ends) are important; deviations in these parameterscaused reduced activity relative to oleic acid (Howling et al., 1972).The position at which the hydroxyl was introduced, however, wasdetermined by the position of the double bond, always being threecarbons distal. Thus, the castor acyl hydroxylase enzyme can produce afamily of different hydroxylated fatty acids depending on theavailability of substrates. Thus, as a matter of convenience, we referto the enzyme throughout as a kappa hydroxylase (rather than an oleatehydroxylase) to indicate the broad substrate specificity.

The castor kappa hydroxylase has many superficial similarities to themicrosomal fatty acyl desaturases (Browse and Somerville, 1991). Inparticular, plants have a microsomal oleate desaturase active at the Δ12position. The substrate of this enzyme (Schmidt et al., 1993) and of thehydroxylase (Bafor et al., 1991) appears to be a fatty acid esterifiedto the sn-2 position of phosphatidylcholine. When oleate is thesubstrate, the modification occurs at the same position (Δ12) in thecarbon chain, and requires the same cofactors, namely electrons fromNADH via cytochrome b₅ and molecular oxygen. Neither enzyme is inhibitedby carbon monoxide (Moreau and Stumpf, 1981), the characteristicinhibitor of cytochrome P450 enzymes.

There do not appear to have been any published biochemical studies ofthe properties of the hydroxylase enzyme(s) in Lesquerella.

Conceptual Basis of the Invention

In U.S. patent application Ser. No. 08/320,982, we described the use ofa cDNA clone from castor for the production of ricinoleic acid intransgenic plants. As noted above, biochemical studies by others hadsuggested that the castor hydroxylase may not have strict specificityfor oleic acid but would also catalyze hydroxylation of other fattyacids such as icosenoic acid (20:1^(cisΔ11)) (Howling et al., 1972).Based on these studies, our previous application Ser. No. 08/320,982noted in Example 2 that the expression of the castor hydroxylase intransgenic plants of species such as Brassica napus and Arabidopsisthaliana that accumulate fatty acids such as icosenoic acid(20:1^(cisΔ11)) and erucic acid (13-docosenoic acid; 22:1^(cisΔ13))would be expected to accumulate some of the hydroxylated derivatives ofthese fatty acids due to the activity of the hydroxylase on these fattyacids. We have now obtained additional direct evidence for such a claimbased on the production of ricinoleic, lesquerolic, densipolic andauricolic fatty acids in transgenic Arabidopsis plants and have includedsuch evidence herein as Example 1.

In Example 3 of the previous application, we taught the various methodsby which the castor hydroxylase clone and sequences derived thereofcould be used to identify other hydroxylase clones from plant speciessuch as Lesquerella fendleri that are known to accumulate hydroxylatedfatty acids in seed oils. In this continuation we have provided anexample of the use of that aspect of the invention for the isolation ofa novel hydroxylase gene from Lesquerella fendleri.

In view of the high degree of sequence similarity between Δ12 fatty aciddesaturases and the castor hydroxylase (van de Loo et al., 1995), thevalidity of claims (e.g., PCT WO 94/11516) for the use of desaturase orhydroxylase genes or sequences derived therefrom for the identificationof genes of identical function from other species must be viewed withskepticism. In this application, we teach a method by which hydroxylasegenes can be distinguished from desaturases and describe methods bywhich Δ12 desaturases can be converted to hydroxylases by themodification of the gene encoding the desaturases. A mechanistic basisfor the similar reaction mechanisms of desaturases and hydroxylases waspresented in the earlier patent application Ser. No. 08/320,982.Briefly, the available evidence suggests that fatty acid desaturaseshave a similar reaction mechanism to the bacterial enzyme methanemonooxygenase which catalyses a reaction involving oxygen-atom transfer(CH₄→CH₃OH) (van de Loo et al., 1993). The cofactor in the hydroxylasecomponent of methane monooxygenase is termed a μ-oxo bridged diironcluster (FeOFe). The two iron atoms of the FeOFe cluster are liganded byprotein-derived nitrogen or oxygen atoms, and are tightly redox-coupledby the covalently-bridging oxygen atom. The FeOFe cluster accepts twoelectrons, reducing it to the diferrous state, before oxygen binding.Upon oxygen binding, it is likely that heterolytic cleavage also occurs,leading to a high valent oxoiron reactive species that is stabilized byresonance rearrangements possible within the tightly coupled FeOFecluster. The stabilized high-valent oxoiron state of methanemonooxygenase is capable of proton extraction from methane, followed byoxygen transfer, giving methanol. The FeOFe cofactor has been shown tobe directly relevant to plant fatty acid modifications by thedemonstration that castor stearoyl-ACP desaturase contains this type ofcofactor (Fox et al., 1993).

On the basis of the foregoing considerations, we hypothesized that thecastor oleate hydroxylase is a structurally modified fatty acyldesaturase, based upon three arguments. The first argument involves thetaxonomic distribution of plants containing ricinoleic acid. Ricinoleicacid has been found in 12 genera of 10 families of higher plants(reviewed in van de Loo et al., 1993). Thus, plants in which ricinoleicacid occurs are found throughout the plant kingdom, yet close relativesof these plants do not contain the unusual fatty acid. This patternsuggests that the ability to synthesize ricinoleic acid has arisen (andbeen lost) several times independently, and is therefore a quite recentdivergence. In other words, the ability to synthesize ricinoleic acidhas evolved rapidly, suggesting that a relatively minor genetic changein the structure of the ancestral enzyme was necessary to accomplish it.

The second argument is that many biochemical properties of castor kappahydroxylase are similar to those of the microsomal desaturases, asdiscussed above (e.g., both preferentially act on fatty acids esterifiedto the sn-2 position of phosphatidylcholine, both use cytochrome b5 asan intermediate electron donor, both are inhibited by cyanide, bothrequire molecular oxygen as a substrate, both are thought to be locatedin the endoplasmic reticulum).

The third argument stems from the discussion of oxygenase cofactorsabove, in which it is suggested that the plant membrane bound fatty aciddesaturases may have a μ-oxo bridged diiron cluster-type cofactor, andthat such cofactors are capable of catalyzing both fatty aciddesaturations and hydroxylations, depending upon the electronic andstructural properties of the protein active site.

Taking these three arguments together, it was hypothesized that kappahydroxylase of castor endosperm is homologous to the microsomal oleateΔ12 desaturase found in all plants. The evidence supporting thishypothesis was disclosed in the previous patent application Ser. No.08/320,982. A number of genes encoding microsomal Δ12 desaturases fromvarious species have recently been cloned (Okuley et al., 1994) andsubstantial information about the structure of these enzymes is nowknown (Shanklin et al. 1994). Hence, in the following invention we teachhow to use structural information about fatty acyl desaturases toisolate kappa hydroxylase genes of this invention. This example teachesthe method by which any carbon-monoxide insensitive plant fatty acylhydroxylase gene can be identified by one skilled in the art.

An unpredicted outcome of our studies on the castor hydroxylase gene intransgenic Arabidopsis plants was the discovery that expression of thehydroxylase leads to increased accumulation of oleic acid in seedlipids. Because of the low nucleotide sequence homology between thecastor hydroxylase and the Δ12-desaturase (about 67%), we consider itunlikely that this effect is due to silencing (also calledsense-suppression or cosuppression) of the expression of the desaturasegene by the hydroxylase gene. Whatever the basis for the effect, thisinvention teaches the use of hydroxylase genes to alter the level offatty acid unsaturation in transgenic plants. On the basis of ahypothesis about the mechanisms of the effect, this invention alsoteaches the use of genetically modified hydroxylase and desaturase genesto achieve directed modification of fatty acid unsaturation levels.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-D show the mass spectra of hydroxy fatty acids standards (FIG.1A, O-TMS-methylricinoleate; FIG. 1B, O-TMS-methyl densipoleate; FIG.1C, O-TMS-methyl-lesqueroleate; and FIG. 1D, O-TMS-methylauricoleate).

FIG. 2 shows the fragmentation pattern of trimethylsilylated methylesters of hydroxy fatty acids.

FIG. 3A shows the gas chromatogram of fatty acids extracted from seedsof wild type Arabidopsis plants. FIG. 3B shows the gas chromatogram offatty acids extracted from seeds of transgenic Arabidopsis plantscontaining the fah12 hydroxylase gene. The numbers indicate thefollowing fatty acids: [1] 16:0; [2] 18:0; [3] 18:1cisΔ9; [4]18:2^(cisΔ9,12); [5] 20:0; [6] 20:1^(cisΔ11); [7] 18:3^(cisΔ9,12,15);[8] 22:1^(cisΔ13); [9] 24:1^(cisΔ13); [10] ricinoleic acid; [11]densipolic acid; [12] lesquerolic acid; [13] auricolic acid.

FIGS. 4A-D show the mass spectra of novel fatty acids found in seeds oftransgenic plants. FIG. 4A shows the mass spectrum of peak 10 from FIG.3B. FIG. 4B shows the mass spectrum of peak 11 from FIG. 3B. FIG. 4Cshows the mass spectrum of peak 12 from FIG. 3B. FIG. 4D shows the massspectrum of peak 13 from FIG. 3B.

FIG. 5 shows the nucleotide sequence of pLesq2 (SEQ ID NO:1).

FIG. 6 shows the nucleotide sequence of pLesq3 (SEQ ID NO:2).

FIG. 7 shows a Northern blot of total RNA from seeds of L. fendleriprobed with pLesq2 or pLesq3. S, indicates RNA is from seeds; L,indicates RNA is from leaves.

FIGS. 8A-B show the nucleotide sequence of genomic clone encodingpLesq-HYD (SEQ ID NO:3), and the deduced amino acid sequence ofhydroxylase enzyme encoded by the gene (SEQ ID NO:4).

FIGS. 9A-B show multiple sequence alignment of deduced amino acidsequences for kappa hydroxylases and microsomal Δ12 desaturases.Abbreviations are: Rcfah12, fah12 hydroxylase gene from R. communis (vande Loo et al., 1995); Lffah12, kappa hydroxylase gene from L. fendleri;Atfad2, fad2 desaturase from Arabidopsis thaliana (Okuley et al., 1994);Gmfad2-1, fad2 desaturase from Glycine max (GenBank accession numberL43920); Gmfad2-2, fad2 desaturase from Glycine max (Genbank accessionnumber L43921); Zmfad2, fad2 desaturase from Zea mays (PCT/US93/09987);Rcfad2, fragment of fad2 desaturase from R. communis (PCT/US93/09987);Bnfad2, fad2 desaturase from Brassica napus (PCT/US93/09987);LFFAH12.AMI, SEQ ID NO:4; FAH12.AMI, SEQ ID NO:S; ATFAD2.AMI, SEQ IDNO:6; BNFAD2.AMI, SEQ ID NO:7; GMFAD2-1.AMI, SEQ ID NO:8; GMFAD2-2.AMI,SEQ ID NO:9; ZMFAD2.AMI, SEQ ID NO:10; and RCFAD2.AMI, SEQ ID NO:11.

FIG. 10 shows a Southern blot of genomic DNA from L. fendleri probedwith pLesq-HYD. E=EcoRI, H=HindIII, X=XbaI.

FIG. 11 shows a map of binary Ti plasmid pSLJ44024.

FIG. 12 shows a map of plasmid pYES2.0

FIG. 13 shows part of a gas chromatogram of derivatized fatty acids fromyeast cells that contain plasmid pLesqYes in which expression of thehydroxylase gene was induced by addition of galactose to the growthmedium. The arrow points to a peak that is not present in uninducedcells. The lower part of the figure is the mass spectrum of the peakindicated by the arrow.

SUMMARY OF THE INVENTION

This invention relates to plant fatty acyl hydroxylases. Methods to useconserved amino acid or nucleotide sequences to obtain plant fatty acylhydroxylases are described. Also described is the use of cDNA clonesencoding a plant hydroxylase to produce a family of hydroxylated fattyacids in transgenic plants.

In a first embodiment, this invention is directed to recombinant DNAconstructs which can provide for the transcription or transcription andtranslation (expression) of the plant kappa hydroxylase sequence. Inparticular, constructs which are capable of transcription ortranscription and translation in plant host cells are preferred. Suchconstructs may contain a variety of regulatory regions includingtranscriptional initiation regions obtained from genes preferentiallyexpressed in plant seed tissue. In a second aspect, this inventionrelates to the presence of such constructs in host cells, especiallyplant host cells which have an expressed plant kappa hydroxylasetherein.

In yet another aspect, this invention relates to a method for producinga plant kappa hydroxylase in a host cell or progeny thereof via theexpression of a construct in the cell. Cells containing a plant kappahydroxylase as a result of the production of the plant kappa hydroxylaseencoding sequence are also contemplated herein.

In another embodiment, this invention relates to methods of using a DNAsequence encoding a plant kappa hydroxylase for the modification of theproportion of hydroxylated fatty acids produced within a cell,especially plant cells. Plant cells having such a modified hydroxylatedfatty acid composition are also contemplated herein.

In a further aspect of this invention, plant kappa hydroxylase proteinsand sequences which are related thereto, including amino acid andnucleic acid sequences, are contemplated. Plant kappa hydroxylaseexemplified herein includes a Lesquerella fendleri fatty acidhydroxylase. This exemplified fatty acid hydroxylase may be used toobtain other plant fatty acid hydroxylases of this invention.

In a further aspect of this invention, a nucleic acid sequence whichdirects the seed specific expression of an associated polypeptide codingsequence is described. The use of this nucleic acid sequence orfragments derived thereof, to obtain seed-specific expression in higherplants of any coding sequence is contemplated herein.

In a further aspect of this invention, the use of genes encoding fattyacyl hydroxylases of this invention are used to alter the amount offatty acid unsaturation of seed lipids. We further envision the use ofgenetically modified hydroxylase and desaturase genes to achievedirected modification of fatty acid unsaturation levels.

DETAILED DESCRIPTION OF THE INVENTION

A genetically transformed plant of the present invention whichaccumulates hydroxylated fatty acids can be obtained by expressing thedouble-stranded DNA molecules described in this application.

A plant fatty acid hydroxylase of this invention includes any sequenceof amino acids, such as a protein, polypeptide or peptide fragment, ornucleic acid sequences encoding such polypeptides, obtainable from aplant source which demonstrates the ability to catalyze the productionof ricinoleic, lesquerolic, hydroxyerucic (16-hydroxydocos-cis-13-enoicacid) or hydroxypalmitoleic (12-hydroxyhexadec-cis-9-enoic) from CoA,ACP or lipid-linked monoenoic fatty acid substrates under plant enzymereactive conditions. By “enzyme reactive conditions” is meant that anynecessary conditions are available in an environment (i.e., such factorsas temperature, pH, lack of inhibiting substances) which will permit theenzyme to function.

Preferential activity of a plant fatty acid hydroxylase toward aparticular fatty acyl substrate is determined upon comparison ofhydroxylated fatty acid product amounts obtained per different fattyacyl substrates. For example, by “oleate preferring” is meant that thehydroxylase activity of the enzyme preparation demonstrates a preferencefor oleate-containing substrates over other substrates. Although theprecise substrate of the castor fatty acid hydroxylase is not known, itis thought to be a monounsaturated fatty acid moiety which is esterifiedto a phospholipid such as phosphatidylcholine. However, it is alsopossible that monounsaturated fatty acids esterified tophosphatidylethanolamine, phosphatidic acid or a neutral lipid such asdiacylglycerol or a Coenzyme-A thioester may also be substrates.

As noted above, significant activity has been observed in radioactivelabelling studies using fatty acyl substrates other than oleate (Howlinget al., 1972) indicating that the substrate specificity is for a familyof related fatty acyl compounds. Because the castor hydroxylaseintroduces hydroxy groups three carbons from a double bond, proximal tothe methyl carbon of the fatty acid, we term the enzyme a kappahydroxylase for convenience. Of particular interest, we envision thatthe castor kappa hydroxylase may be used for production of12-hydroxy-9-octadecenoic acid (ricinoleate), 12-hydroxy-9-hexadecenoicacid, 14-hydroxy-11-eicosenoic acid, 16-hydroxy-13-docosanoic acid,9-hydroxy-6-octadecenoic acid by expression in plants species whichproduce the non-hydroxylated precursors. We also envision production ofadditionally modified fatty acids such as12-hydroxy-9,15-octadecadienoic acid that result from desaturation ofhydroxylated fatty acids (e.g., 12-hydroxy-9-octadecenoic acid in thisexample).

We also envision that future advances in the genetic engineering ofplants will lead to production of substrate fatty acids, such asicosenoic acid esters, and palmitoleic acid esters in plants that do notnormally accumulate such fatty acids. We envision that the inventiondescribed herein may be used in conjunction with such futureimprovements to produce hydroxylated fatty acids of this invention inany plant species that is amenable to directed genetic modification.Thus, the applicability of this invention is not limited in ourconception only to those species that currently accumulate suitablesubstrates.

As noted above, a plant kappa hydroxylase of this invention will displayactivity towards various fatty acyl substrates. During biosynthesis oflipids in a plant cell, fatty acids are typically covalently bound toacyl carrier protein (ACP), coenzyme A (CoA) or various cellular lipids.Plant kappa hydroxylases which display preferential activity towardlipid-linked acyl substrate are especially preferred because they arelikely to be closely associated with normal pathway of storage lipidsynthesis in immature embryos. However, activity toward acyl-CoAsubstrates or other synthetic substrates, for example, is alsocontemplated herein.

Other plant kappa hydroxylases are obtainable from the specificexemplified sequences provided herein. Furthermore, it will be apparentthat one can obtain natural and synthetic plant kappa hydroxylasesincluding modified amino acid sequences and starting materials forsynthetic-protein modeling from the exemplified plant kappa hydroxylaseand from plant kappa hydroxylases which are obtained through the use ofsuch exemplified sequences. Modified amino acid sequences includesequences which have been mutated, truncated, increased and the like,whether such sequences were partially or wholly synthesized. Sequenceswhich are actually purified from plant preparations or are identical orencode identical proteins thereto, regardless of the method used toobtain the protein or sequence, are equally considered naturallyderived.

Thus, one skilled in the art will readily recognize that antibodypreparations, nucleic acid probes (DNA and RNA) and the like may beprepared and used to screen and recover “homologous” or “related” kappahydroxylases from a variety of plant sources. Typically, nucleic acidprobes are labeled to allow detection, preferably with radioactivityalthough enzymes or other methods may also be used. For immunologicalscreening methods, antibody preparations either monoclonal or polyclonalare utilized. Polyclonal antibodies, although less specific, typicallyare more useful in gene isolation. For detection, the antibody islabeled using radioactivity or any one of a variety of secondantibody/enzyme conjugate systems that are commercially available.

Homologous sequences are found when there is an identity of sequence andmay be determined upon comparison of sequence information, nucleic acidor amino acid, or through hybridization reactions between a known kappahydroxylase and a candidate source. Conservative changes, such asGlu/Asp, Val/Ile, Ser/Thr, Arg/Lys and Gln/Asn may also be considered indetermining sequence homology. Typically, a lengthy nucleic acidsequence may show as little as 50-60% sequence identity, and morepreferably at least about 70% sequence identity, between the targetsequence and the given plant kappa hydroxylase of interest excluding anydeletions which may be present, and still be considered related. Aminoacid sequences are considered homologous by as little as 25% sequenceidentity between the two complete mature proteins. (See generally,Doolittle, R. F., OF URFS and ORFS, University Science Books, Calif.,1986.)

A genomic or other appropriate library prepared from the candidate plantsource of interest may be probed with conserved sequences from the plantkappa hydroxylase to identify homologously related sequences. Use of anentire cDNA or other sequence may be employed if shorter probe sequencesare not identified. Positive clones are then analyzed by restrictionenzyme digestion and/or sequencing. When a genomic library is used, oneor more sequences may be identified providing both the coding region, aswell as the transcriptional regulatory elements of the kappa hydroxylasegene from such plant source. Probes can also be considerably shorterthan the entire sequence. Oligonucleotides may be used, for example, butshould be at least about 10, preferably at least about 15, morepreferably at least 20 nucleotides in length. When shorter lengthregions are used for comparison, a higher degree of sequence identity isrequired than for longer sequences. Shorter probes are oftenparticularly useful for polymerase chain reactions (PCR), especiallywhen highly conserved sequences can be identified (See Gould, et al.,1989 for examples of the use of PCR to isolate homologous genes fromtaxonomically diverse species).

When longer nucleic acid fragments are employed (>100 bp) as probes,especially when using complete or large cDNA sequences, one would screenwith low stringencies (for example, 40-50° C. below the meltingtemperature of the probe) in order to obtain signal from the targetsample with 20-50% deviation, i.e., homologous sequences. (Beltz, et al.1983).

In a preferred embodiment, a plant kappa hydroxylase of this inventionwill have at least 60% overall amino acid sequence similarity with theexemplified plant kappa hydroxylase. In particular, kappa hydroxylaseswhich are obtainable from an amino acid or nucleic acid sequence of acastor or lesquerella kappa hydroxylase are especially preferred. Theplant kappa hydroxylases may have preferential activity toward longer orshorter chain fatty acyl substrates. Plant fatty acyl hydroxylaseshaving oleate-12-hydroxylase activity and eicosenoate-14-hydroxylaseactivity are both considered homologously related proteins because of invitro evidence (Howling et al., 1972), and evidence disclosed herein,that the castor kappa hydroxylase will act on both substrates.Hydroxylated fatty acids may be subject to further enzymaticmodification by other enzymes which are normally present or areintroduced by genetic engineering methods. For example,14-hydroxy-11,17-eicosadienoic acid, which is present in someLesquerella species (Smith 1985), is thought to be produced bydesaturation of 14-hydroxy-11-eicosenoic acid.

Again, not only can gene clones and materials derived thereof be used toidentify homologous plant fatty acyl hydroxylases, but the resultingsequences obtained therefrom may also provide a further method to obtainplant fatty acyl hydroxylases from other plant sources. In particular,PCR may be a useful technique to obtain related plant fatty acylhydroxylases from sequence data provided herein. One skilled in the artwill be able to design oligonucleotide probes based upon sequencecomparisons or regions of typically highly conserved sequence. Ofspecial interest are polymerase chain reaction primers based on theconserved regions of amino acid sequence between the castor kappahydroxylase and the L. fendleri hydroxylase (SEQ ID NO:4). Detailsrelating to the design and methods for a PCR reaction using these probesare described more fully in the examples.

It should also be noted that the fatty acyl hydroxylases of a variety ofsources can be used to investigate fatty acid hydroxylation events in awide variety of plant and in vivo applications. Because all plantssynthesize fatty acids via a common metabolic pathway, the study and/orapplication of one plant fatty acid hydroxylase to a heterologous planthost may be readily achieved in a variety of species.

Once the nucleic acid sequence is obtained, the transcription, ortranscription and translation (expression), of the plant fatty acylhydroxylases in a host cell is desired to produce a ready source of theenzyme and/or modify the composition of fatty acids found therein in theform of free fatty acids, esters (particularly esterified toglycerolipids or as components of wax esters), estolides, or ethers.Other useful applications may be found when the host cell is a planthost cell, in vitro and in vivo. For example, by increasing the amountof an kappa hydroxylase available to the plant, an increased percentageof ricinoleate or lesqueroleate (14-hydroxy-11-eicosenoic acid) may beprovided.

Kappa Hydroxylase

By this invention, a mechanism for the biosynthesis of ricinoleic acidin plants is demonstrated. Namely, that a specific plant kappahydroxylase having preferential activity toward fatty acyl substrates isinvolved in the accumulation of hydroxylated fatty acids in at leastsome plant species. The use of the terms ricinoleate or ricinoleic acid(or lesqueroleate or lesquerolic acid, densipoleate etc.) is intended toinclude the free acids, the ACP and CoA esters, the salts of theseacids, the glycerolipid esters (particularly the triacylglycerolesters), the wax esters, the estolides and the ether derivatives ofthese acids.

The determination that plant fatty acyl hydroxylases are active in thein vivo production of hydroxylated fatty acids suggests severalpossibilities for plant enzyme sources. In fact, hydroxylated fattyacids are found in some natural plant species in abundance. For example,three hydroxy fatty acids related to ricinoleate occur in major amountsin seed oils from various Lesquerella species. Of particular interest,lesquerolic acid is a 20 carbon homolog of ricinoleate with twoadditional carbons at the carboxyl end of the chain (Smith 1985). Othernatural plant sources of hydroxylated fatty acids include but are notlimited to seeds of the Linum genus, seeds of Wrightia species,Lycopodium species, Strophanthus species, Convolvulaces species,Calendula species and many others (van de Loo et al., 1993).

Plants having significant presence of ricinoleate or lesqueroleate ordesaturated other or modified derivatives of these fatty acids arepreferred candidates to obtain naturally-derived kappa hydroxylases. Forexample, Lesquerella densipila contains a diunsaturated 18 carbon fattyacid with a hydroxyl group (van de Loo et al., 1993) that is thought tobe produced by an enzyme that is closely related to the castor kappahydroxylase, according to the theory on which this invention is based.In addition, a comparison between kappa hydroxylases and between plantfatty acyl hydroxylases which introduce hydroxyl groups at positionsother than the 12-carbon of oleate or the 14-carbon of lesqueroleate oron substrates other than oleic acid and icosenoic acid may yieldinsights for protein modeling or other modifications to create synthetichydroxylases as discussed above. For example, on the basis ofinformation gained from structural comparisons of the Δ12 desaturasesand the kappa hydroxylase, we envision making genetic modifications inthe structural genes for Δ12 desaturases that convert these desaturasesto kappa-hydroxylases. We also envision making changes in Δ15hydroxylases that convert these to hydroxylases with comparablesubstrate specificity to the desaturases (e.g., conversion of18:2^(Δ9,12) to 15OH-18:2^(Δ9,12). Since the difference between ahydroxylase and a desaturases concerns the disposition of one proton, weenvision that by systematically changing the charged groups in theregion of the enzyme near the active site, we can effect this change.

Especially of interest are fatty acyl hydroxylases which demonstrateactivity toward fatty acyl substrates other than oleate, or whichintroduce the hydroxyl group at a location other than the C12 carbon. Asdescribed above, other plant sources may also provide sources for theseenzymes through the use of protein purification, nucleic acid probes,antibody preparations, protein modeling, or sequence comparisons, forexample, and of special interest are the respective amino acid andnucleic acid sequences corresponding to such plant fatty acylhydroxylases. Also as previously described, once a nucleic acid sequenceis obtained for the given plant hydroxylase, further plant sequences maybe compared and/or probed to obtain homologously related DNA sequencesthereto and so on.

Genetic Engineering Applications

As is well known in the art, once a cDNA clone encoding a plant kappahydroxylase is obtained, it may be used to obtain its correspondinggenomic nucleic acid sequences thereto.

The nucleic acid sequences which encode plant kappa hydroxylases may beused in various constructs, for example, as probes to obtain furthersequences from the same or other species. Alternatively, these sequencesmay be used in conjunction with appropriate regulatory sequences toincrease levels of the respective hydroxylase of interest in a host cellfor the production of hydroxylated fatty acids or study of the enzyme invitro or in vivo or to decrease or increase levels of the respectivehydroxylase of interest for some applications when the host cell is aplant entity, including plant cells, plant parts (including but notlimited to seeds, cuttings or tissues) and plants.

A nucleic acid sequence encoding a plant kappa hydroxylase of thisinvention may include genomic, cDNA or mRNA sequence. By “encoding” ismeant that the sequence corresponds to a particular amino acid sequenceeither in a sense or anti-sense orientation. By “recombinant” is meantthat the sequence contains a genetically engineered modification throughmanipulation via mutagenesis, restriction enzymes, and the like. A cDNAsequence may or may not encode pre-processing sequences, such as transitor signal peptide sequences. Transit or signal peptide sequencesfacilitate the delivery of the protein to a given organelle and arefrequently cleaved from the polypeptide upon entry into the organelle,releasing the “mature” sequence. The use of the precursor DNA sequenceis preferred in plant cell expression cassettes.

Furthermore, as discussed above the complete genomic sequence of theplant kappa hydroxylase may be obtained by the screening of a genomiclibrary with a probe, such as a cDNA probe, and isolating thosesequences which regulate expression in seed tissue. In this manner, thetranscription and translation initiation regions, introns, and/ortranscript termination regions of the plant kappa hydroxylase may beobtained for use in a variety of DNA constructs, with or without thekappa hydroxylase structural gene. Thus, nucleic acid sequencescorresponding to the plant kappa hydroxylase of this invention may alsoprovide signal sequences useful to direct transport into an organelle 5′upstream non-coding regulatory regions (promoters) having useful tissueand timing profiles, 3′ downstream non-coding regulatory region usefulas transcriptional and translational regulatory regions and may lendinsight into other features of the gene.

Once the desired plant kappa hydroxylase nucleic acid sequence isobtained, it may be manipulated in a variety of ways. Where the sequenceinvolves non-coding flanking regions, the flanking regions may besubjected to resection, mutagenesis, etc. Thus, transitions,transversions, deletions, and insertions may be performed on thenaturally occurring sequence. In addition, all or part of the sequencemay be synthesized. In the structural gene, one or more codons may bemodified to provide for a modified amino acid sequence, or one or morecodon mutations may be introduced to provide for a convenientrestriction site or other purpose involved with construction orexpression. The structural gene may be further modified by employingsynthetic adapters, linkers to introduce one or more convenientrestriction sites, or the like.

The nucleic acid or amino acid sequences encoding a plant kappahydroxylase of this invention may be combined with other non-native, or“heterologous”, sequences in a variety of ways. By “heterologous”sequences is meant any sequence which is not naturally found joined tothe plant kappa hydroxylase, including, for example, combination ofnucleic acid sequences from the same plant which are not naturally foundjoined together.

The DNA sequence encoding a plant kappa hydroxylase of this inventionmay be employed in conjunction with all or part of the gene sequencesnormally associated with the kappa hydroxylase. In its component parts,a DNA sequence encoding kappa hydroxylase is combined in a DNA constructhaving, in the 5′ to 3′ direction of transcription, a transcriptioninitiation control region capable of promoting transcription andtranslation in a host cell, the DNA sequence encoding plant kappahydroxylase and a transcription and translation termination region.

Potential host cells include both prokaryotic and eukaryotic cells. Ahost cell may be unicellular or found in a multicellular differentiatedor undifferentiated organism depending upon the intended use. Cells ofthis invention may be distinguished by having a plant kappa hydroxylaseforeign to the wild-type cell present therein, for example, by having arecombinant nucleic acid construct encoding a plant kappa hydroxylasetherein.

Depending upon the host, the regulatory regions will vary, includingregions from viral, plasmid or chromosomal genes, or the like. Forexpression in prokaryotic or eukaryotic microorganisms, particularlyunicellular hosts, a wide variety of constitutive or regulatablepromoters may be employed. Expression in a microorganism can provide aready source of the plant enzyme. Among transcriptional initiationregions which have been described are regions from bacterial and yeasthosts, such as E. coli, B. subtilis, Saccharomyces cerevisiae, includinggenes such as beta-galactosidase, T7 polymerase, tryptophan E and thelike.

For the most part, the constructs will involve regulatory regionsfunctional in plants which provide for modified production of plantkappa hydroxylase with resulting modification of the fatty acidcomposition. The open reading frame, coding for the plant kappahydroxylase or functional fragment thereof will be joined at its 5′ endto a transcription initiation regulatory region such as the wild-typesequence naturally found 5′ upstream to the kappa hydroxylase structuralgene. Numerous other transcription initiation regions are availablewhich provide for a wide variety of constitutive or regulatable, e.g.,inducible, transcription of the structural gene functions.

Among transcriptional initiation regions used for plants are suchregions associated with the structural genes such as for nopaline andmannopine synthases, or with napin, soybean β-conglycinin, oleosin, 12Sstorage protein, the cauliflower mosaic virus 35S promoters and thelike. The transcription/translation initiation regions corresponding tosuch structural genes are found immediately 5′ upstream to therespective start codons.

In embodiments wherein the expression of the kappa hydroxylase proteinis desired in a plant host, the use of all or part of the complete plantkappa hydroxylase gene is desired; namely all or part of the 5′ upstreamnon-coding regions (promoter) together with the structural gene sequenceand 3′ downstream non-coding regions may be employed. If a differentpromoter is desired, such as a promoter native to the plant host ofinterest or a modified promoter, i.e., having transcription initiationregions derived from one gene source and translation initiation regionsderived from a different gene source, including the sequence encodingthe plant kappa hydroxylase of interest, or enhanced promoters, such asdouble 35S CaMV promoters, the sequences may be joined together usingstandard techniques.

For such applications when 5′ upstream non-coding regions are obtainedfrom other genes regulated during seed maturation, those preferentiallyexpressed in plant embryo tissue, such as transcription initiationcontrol regions from the B. napus napin gene, or the Arabidopsis 12Sstorage protein, or soybean β-conglycinin (Bray et al., 1987), or the L.fendleri kappa hydroxylase promoter described herein are desired.Transcription initiation regions which are preferentially expressed inseed tissue, i.e., which are undetectable in other plant parts, areconsidered desirable for fatty acid modifications in order to minimizeany disruptive or adverse effects of the gene product.

Regulatory transcript termination regions may be provided in DNAconstructs of this invention as well. Transcript termination regions maybe provided by the DNA sequence encoding the plant kappa hydroxylase ora convenient transcription termination region derived from a differentgene source, for example, the transcript termination region which isnaturally associated with the transcript initiation region. Where thetranscript termination region is from a different gene source, it willcontain at least about 0.5 kb, preferably about 1-3 kb of sequence 3′ tothe structural gene from which the termination region is derived.

Plant expression or transcription constructs having a plant kappahydroxylase as the DNA sequence of interest for increased or decreasedexpression thereof may be employed with a wide variety of plant life,particularly, plant life involved in the production of vegetable oilsfor edible and industrial uses. Most especially preferred are temperateoilseed crops. Plants of interest include, but are not limited torapeseed (Canola and high erucic acid varieties), Crambe, Brassicajuncea, Brassica nigra, meadowfoam, flax, sunflower, safflower, cotton,Cuphea, soybean, peanut, coconut and oil palms and corn. An importantcriterion in the selection of suitable plants for the introduction onthe kappa hydroxylase is the presence in the host plant of a suitablesubstrate for the hydroxylase. Thus, for example, production ofricinoleic acid will be best accomplished in plants that normally havehigh levels of oleic acid in seed lipids. Similarly, production oflesquerolic acid will best be accomplished in plants that have highlevels of icosenoic acid in seed lipids.

Depending on the method for introducing the recombinant constructs intothe host cell, other DNA sequences may be required. Importantly, thisinvention is applicable to dicotyledons and monocotyledons species alikeand will be readily applicable to new and/or improved transformation andregulation techniques. The method of transformation is not critical tothe current invention; various methods of plant transformation arecurrently available. As newer methods are available to transform crops,they may be directly applied hereunder. For example, many plant speciesnaturally susceptible to Agrobacterium infection may be successfullytransformed via tripartite or binary vector methods of Agrobacteriummediated transformation. In addition, techniques of microinjection, DNAparticle bombardment, electroporation have been developed which allowfor the transformation of various monocot and dicot plant species.

In developing the DNA construct, the various components of the constructor fragments thereof will normally be inserted into a convenient cloningvector which is capable of replication in a bacterial host, e.g., E.coli. Numerous vectors exist that have been described in the literature.After each cloning, the plasmid may be isolated and subjected to furthermanipulation, such as restriction, insertion of new fragments, ligation,deletion, insertion, resection, etc., so as to tailor the components ofthe desired sequence. Once the construct has been completed, it may thenbe transferred to an appropriate vector for further manipulation inaccordance with the manner of transformation of the host-cell.

Normally, included with the DNA construct will be a structural genehaving the necessary regulatory regions for expression in a host andproviding for selection of transformant cells. The gene may provide forresistance to a cytotoxic agent, e.g., antibiotic, heavy metal, toxin,etc., complementation providing prototropy to an auxotrophic host, viralimmunity or the like. Depending upon the number of different hostspecies the expression construct or components thereof are introduced,one or more markers may be employed, where different conditions forselection are used for the different hosts.

It is noted that the degeneracy of the DNA code provides that some codonsubstitutions are permissible of DNA sequences without any correspondingmodification of the amino acid sequence.

As mentioned above, the manner in which the DNA construct is introducedinto the plant host is not critical to this invention. Any method whichprovides for efficient transformation may be employed. Various methodsfor plant cell transformation include the use of Ti- or Ri-plasmids,microinjection, electroporation, infiltration, imbibition, DNA particlebombardment, liposome fusion, DNA bombardment or the like. In manyinstances, it will be desirable to have the construct bordered on one orboth sides of the T-DNA, particularly having the left and right borders,more particularly the right border. This is particularly useful when theconstruct uses A. tumefaciens or A. rhizogenes as a mode fortransformation, although the T-DNA borders may find use with other modesof transformation.

Where Agrobacterium is used for plant cell transformation, a vector maybe used which may be introduced into the Agrobacterium host forhomologous recombination with T-DNA or the Ti- or Ri-plasmid present inthe Agrobacterium host. The Ti- or Ri-plasmid containing the T-DNA forrecombination may be armed (capable of causing gall formation) ordisarmed (incapable of causing gall), the latter being permissible, solong as the vir genes are present in the transformed Agrobacterium host.The armed plasmid can give a mixture of normal plant cells and gall.

In some instances where Agrobacterium is used as the vehicle fortransforming plant cells, the expression construct bordered by the T-DNAborder(s) will be inserted into a broad host spectrum vector, therebeing broad host spectrum vectors described in the literature. Commonlyused is pRK2 or derivatives thereof. See, for example, Ditta et al.,(1980), which is incorporated herein by reference. Included with theexpression construct and the T-DNA will be one or more markers, whichallow for selection of transformed Agrobacterium and transformed plantcells. A number of markers have been developed for use with plant cells,such as resistance to kanamycin, the aminoglycoside G418, hygromycin, orthe like. The particular marker employed is not essential to thisinvention, one or another marker being preferred depending on theparticular host and the manner of construction.

For transformation of plant cells using Agrobacterium, explants may becombined and incubated with the transformed Agrobacterium for sufficienttime for transformation, the bacteria killed, and the plant cellscultured in an appropriate selective medium. Once callus forms, shootformation can be encouraged by employing the appropriate plant hormonesin accordance with known methods and the shoots transferred to rootingmedium for regeneration of plants. The plants may then be grown to seedand the seed used to establish repetitive generations and for isolationof vegetable oils.

Using Hydroxylase Genes to Alter the Activity of Fatty Acid Desaturases

A widely acknowledged goal of current efforts to improve the nutritionalquality of edible plant oils, or to facilitate industrial applicationsof plant oils, is to alter the level of desaturation of plant storagelipids (Topfer et al., 1995). In particular, in many crop species it isconsidered desirable to reduce the level of polyunsaturation of storagelipids and to increase the level of oleic acid. The precise amount ofthe various fatty acids in a particular plant oil varies with theintended application. Thus, it is desirable to have a robust method thatwill permit genetic manipulation of the level of unsaturation to anydesired level.

Substantial progress has recently been made in the isolation of genesencoding plant fatty acid desaturases (reviewed in Topfer et al., 1995).These genes have been introduced into various plant species and used toalter the level of fatty acid unsaturation in one of three ways. First,the genes can be placed under transcriptional control of a strongpromoter so that the amount of the corresponding enzyme is increased. Insome cases this leads to an increase in the amount of the fatty acidthat is the product of the reaction catalyzed by the enzyme. Forexample, Arondel et al. (1992) increased the amount of linolenic acid(18:3) in tissues of transgenic Arabidopsis plants by placing theendoplasmic reticulum-localized fad3 gene under transcriptional controlof the strong constitutive cauliflower mosaic virus 35S promoter.

A second method of using cloned genes to alter the level of fatty acidunsaturation is to cause transcription of all or part of a gene intransgenic tissues so that the transcripts have an antisense orientationrelative to the normal mode of transcription. This has been used by anumber of laboratories to reduce the level of expression of one or moredesaturase genes that have significant nucleotide sequence homology tothe gene used in the construction of the antisense gene (reviewed inTopfer et al.). For instance, antisense repression of the oleateΔ12-desaturase in transgenic rapeseed resulted in a strong increase inoleic-acid content (cf., Topfer et al., 1995).

A third method for using cloned genes to alter fatty acid desaturationis to exploit the phenomenon of cosuppression or “gene-silencing”(Matzke et al., 1995). Although the mechanisms responsible for genesilencing are not known in any detail, it has frequently been observedthat in transgenic plants, expression of an introduced gene leads toinactivation of homologous endogenous genes.

For example, high-level sense expression of the Arabidopsis fad8 gene,which encodes a chloroplast-localized Δ15-desaturase, in transgenicArabidopsis plants caused suppression of the endogenous copy of the fad8gene and the homologous fad7 gene (which encodes an isozyme of the fad8gene) (Gibson et al., 1994). The fad7 and fad8 genes are only 76%identical at the nucleotide level. At the time of publication, thisexample represented the most divergent pair of plant genes for whichcosuppression had been observed.

In view of previous evidence concerning the relatively high level ofnucleotide sequence homology required to obtain cosuppression, it is notobvious to one skilled in the art that sense expression in transgenicplants of the castor fatty acyl hydroxylase of this invention wouldsignificantly alter the amount of unsaturation of storage lipids.

However, we have established that fatty acyl hydroxylase genes can beused for this purpose as taught in Example 4 of this invention. Ofparticular importance, this invention teaches the use of fatty acylhydroxylase genes to increase the proportion of oleic acid in transgenicplant tissues. The mechanism by which expression of the gene exerts thiseffect is not known but may be due to one of several possibilities whichare elaborated upon in Example 4.

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are included forpurposes of illustration only and are not intended to limit the presentinvention.

EXAMPLES

In the experimental disclosure which follows, all temperatures are givenin degrees centigrade (°), weights are given in grams (g), milligram(mg) or micrograms (μg), concentrations are given as molar (M),millimolar (mM) or micromolar (μM) and all volumes are given in liters(l), microliters (μl) or milliliters (ml), unless otherwise indicated.

Example 1 Production of Novel-Hydroxylated Fatty Acids in ArabidopsisThaliana Overview

The kappa hydroxylase encoded by the previously described fah12 genefrom Castor (U.S. patent application Ser. No. 08/320,982 was used toproduce ricinoleic acid, lesquerolic acid, densipolic acid and auricolicacid in transgenic Arabidopsis plants. This example specificallydiscloses the method taught in Example 2 of U.S. patent application Ser.No. 08/320,982.

Production of Transgenic Plants

A variety of methods have been developed to insert a DNA sequence ofinterest into the genome of a plant host to obtain the transcription andtranslation of the sequence to effect phenotypic changes. The followingmethods represent only one of many equivalent means of producingtransgenic plants and causing expression of the hydroxylase gene.

Arabidopsis plants were transformed, by Agrobacterium-mediatedtransformation, with the kappa hydroxylase encoded by the Castor fah12gene on binary Ti plasmid pB6. This plasmid was previously used totransform Nicotiana tabacum for the production of ricinoleic acid (U.S.patent application Ser. No. 08/320,982).

Inoculums of Agrobacterium tumefaciens strain GV3101 containing binaryTi plasmid pB6 were plated on L-broth plates containing, 50 μg/mlkanamycin and incubated for 2 days at 30° C. Single colonies were usedto inoculate large liquid cultures (L-broth medium with 50 mg/lrifampicin, 110 mg/l gentamycin and 200 mg/l kanamycin) to be used forthe transformation of Arabidopsis plants.

Arabidopsis plants were transformed by the in planta transformationprocedure essentially as described by Bechtold et al., (1993). Cells ofA. tumefaciens GV3101(pB6) were harvested from liquid cultures bycentrifugation, then resuspended in infiltration medium atOD₆₀₀=0.8-(Infiltration medium was Murashige and Skoog macro andmicronutrient medium (Sigma Chemical Co., St. Louis, Mo.) containing 10mg/l 6-benzylaminopurine and 5% glucose). Batches of 12-15 plants weregrown for 3 to 4 weeks in natural light at a mean daily temperature ofapproximately 25° C. in 3.5 inch pots containing soil. The intact plantswere immersed in the bacterial suspension then transferred to a vacuumchamber and placed under 600 mm of vacuum produced by a laboratoryvacuum pump until tissues appeared uniformly water-soaked (approximately10 min). The plants were grown at 25° C. under continuous light (100μmol m⁻² s⁻¹ irradiation in the 400 to 700 nm range) for four weeks. Theseeds obtained from all the plants in a pot were harvested as one batch.The seeds were sterilized by sequential treatment for 2 min with ethanolfollowed by 10 min in a mixture of household bleach (Chlorox), water andTween-80 (50%, 50%, 0.05%) then rinsed thoroughly with sterile water.The seeds were plated at high density (2000 to 4000 per plate) ontoagar-solidified medium in 100 mm petri plates containing ½× Murashigeand Skoog salts medium enriched with B5 vitamins (Sigma Chemical Co.,St. Louis, Mo.) and containing kanamycin at 50 mg/l. After incubationfor 48 h at 4° C. to stimulate germination, seedlings were grown for aperiod of seven days until transformants were clearly identifiable ashealthy green seedlings against a background of chlorotickanamycin-sensitive seedlings. The transformants were transferred tosoil for two weeks before leaf tissue could be used for DNA and lipidanalysis. More than 20 transformants were obtained.

DNA was extracted from young leaves from transformants to verify thepresence of an intact fah12 gene. The presence of the transgene in anumber of the putative transgenic lines was verified by using thepolymerase chain reaction to amplify the insert from pB6. The primersused were HF2=GCTCTTTTGTGCGCTCATTC (SEQ ID NO:12) andHR1=CGGTACCAGAAAACGCCTTG (SEQ ID NO:13), which were designed to allowthe amplification of a 700 bp fragment. Approximately 100 ng of genomicDNA was added to a solution containing 25 pmol of each primer, 1.5 U Taqpolymerase (Boehringer Manheim), 200 uM of dNTPs, 50 mM KCl, 10 mMTris.Cl (pH 9), 0.1% (v/v) Triton X-100, 1.5 mM MgCl₂, 3% (v/v)formamide, to a final volume of 50 μl.

Amplifications conditions were: 4 min denaturation step at 94° C.,followed by 30 cycles of 92° C. for 1 min, 55° C. for 1 min, 72° C. for2 min. A final extension step closed the program at 72° C. for 5 min.Transformants could be positively identified after visualization of acharacteristic 1 kb amplified fragment on an ethidium bromide stainedagarose gel. All transgenic lines tested gave a PCR product of a sizeconsistent with the expected genotype, confirming that the lines were,indeed, transgenic. All further-experiments were done with threerepresentative transgenic lines of the wild type designated as 1-3, 4D,7-4 and one transgenic line of the fad2 mutant line JB12. The transgenicJB12 line was included in order to test whether the increasedaccumulation of oleic acid in this mutant would have an effect on theamount of ricinoleic acid that accumulated in the transgenic plants.

Analysis of Transgenic Plants

Leaves and seeds from fah12 transgenic Arabidopsis plants were analyzedfor the presence of hydroxylated fatty acids using gas chromatography.Lipids were extracted from 100-200 mg leaf tissue or 50 seeds. Fattyacid methyl esters (FAMES) were prepared by placing tissue in 1.5 ml of1.0 M methanolic HCl (Supelco Co.) in a 13×100 mm glass screw-cap tubecapped with a teflon-lined cap and heated to 80° C. for 2 hours. Uponcooling, 1 ml petroleum ether was added and the FAMES removed byaspirating off the ether phase which was then dried under a nitrogenstream in a glass tube. One hundred μl of N,O-bis(Trimethylsilyl)trifluoroacetamide (BSTFA; Pierce Chemical Co) and 200 μl acetonitrilewas added to derivatize the hydroxyl groups. The reaction was carriedout at 70° C. for 15 min. The products were dried under nitrogen,redissolved in 100 μl chloroform and transferred to a gas chromatographvial. Two μl of each sample were analyzed on a SP2340 fused silicacapillary column (30 m, 0.75 mm ID, 0.20 mm film, Supelco), using aHewlett-Packard 5890 II series Gas Chromatograph. The samples were notsplit, the temperature program was 195° C. for 18 min, increased to 230°C. at 25° C./min, held at 230° C. for 5 min then down to 195° C. at 25°C./min., and flame ionization detectors were used.

The chromatographic elution time of methyl esters and O-TMS derivativesof ricinoleic acid, lesquerolic acid and auricolic acid was establishedby GC-MS of lipid samples from seeds of L. fendleri and comparison topublished chromatograms of fatty acids from this species (Carlson etal., 1990). A O-TMS-methyl-ricinoleate standard was prepared fromricinoleic acid obtained from Sigma Chemical Co (St, Louis, Mo.).O-TMS-methyl-lesqueroleate and O-TMS-methyl-ricinoleate standards wereprepared from triacylglycerols purified from seeds of L. fendleri. Themass spectrum of O-TMS-methyl-ricinoleate, O-TMS-methyl-densipoleate,O-TMS-methyl-lesqueroleate, and O-TMS-methyl-auricoleate are shown inFIGS. 1A-D, respectively. The structures of the characteristic ionsproduced during mass spectrometry of these derivatives are shown in FIG.2.

Lipid extracted from transgenic tissues were analyzed by gaschromatography and mass spectrometry for the presence of hydroxylatedfatty acids. As a matter of reference, the average fatty acidcomposition of leaves in Arabidopsis wild type and fad2 mutant lines wasreported by Miquel and Browse (1992). Gas chromatograms of methylatedand silylated fatty acids from seeds of wild type and a fah12 transgenicwild type plant are shown in FIGS. 3A and 3B, respectively. The profilesare very similar except for the presence of three small but distinctpeaks at 14.3, 15.9 and 18.9 minutes. A very small peak at 20.15 min wasalso evident. The elution time of the peaks at 14.3 and 18.9 mincorresponded precisely to that of comparably prepared ricinoleic andlesquerolic standards, respectively. No significant differences wereobserved in lipid extracts from leaves or roots of the wild type and thefah12 transgenic wild type lines (Table 1). Thus, in spite of the factthat the fah12 gene is expressed throughout the plant, we observedeffects on fatty acid composition only in seed tissue. A similarobservation was described previously for transgenic fah12 tobacco inpatent application Ser. No. 08/320,982.

TABLE 1 Fatty acid composition of lipids from transgenic and wild typeArabidopsis. Fatty Seed Leaf Root acid WT FAH12/WT FAH12/fad2 JB12 WTFAH12/WT WT FAH12/WT 16:0 8.5 8.2 6.4 6.1 16.5 17.5 23.9 24.9 16:3 0 0 00 10.1 9.8 0 0 18:0 3.2 3.5 2.9 3.5 1.3 1.2 2.0 1.9 18:1 15.4 26.3 43.447.8 2.4 3.4 5.4 3.2 18:2 27.0 21.4 10.2 7.2 15.1 14.0 32.2 29.4 18:322.0 16.6 — 9.7 36.7 36.0 26.7 30.6 20:1 14.0 14.3 — 13.1 0 0 0 0 22:12.0 1.0 0.5 0.5 0 0 0 0 24:1 2.5 1.7 2.0 1.6 0 0 0 0 18:1-OH 0 0.4 0.3 00 0 0 0 18:2-OH 0 0.4 0.3 0 0 0 0 0 20:1-OH 0 0.2 0.1 0 0 0 0 0 20:2-OH0 0.1 0.1 0 0 0 0 0 The values are the means obtained from analysis ofsamples from three independent transgenic lines, or three independentsamples of wild type and fad2 lines.

In order to confirm that the observed new peaks in the transgenic linescorresponded to derivatives of ricinoleic, lesquerolic, densipolic andauricolic acids, mass spectrometry was used. The fatty acid derivativeswere resolved by gas chromatography as described above except that aHewlett-Packard 5971 series mass selective detector was used in place ofthe flame ionization detector used in the previous experiment. Thespectra of the four new peaks in FIG. 3B (peak numbers 10, 11, 12 and13) are shown in FIGS. 4A-D, respectively. Comparison of the spectrumobtained for the standards with that obtained for the four peaks fromthe transgenic lines confirms the identity of the four new peaks. On thebasis of the three characteristic peaks at M/Z 187, 270 and 299, peak 10is unambiguously identified as O-TMS-methylricinoleate. On the basis ofthe three characteristic peaks at M/Z 185, 270 and 299, peak 11 isunambiguously identified as O-TMS-methyldensipoleate. On the basis ofthe three characteristic peaks at M/Z 187, 298 and 327, peak 12 isunambiguously identified as O-TMS-methyllesqueroleate. On the basis ofthe three characteristic peaks at M/Z 185, 298 and 327, peak 13 isunambiguously identified as O-TMS-methylauricoleate.

These results unequivocally demonstrate the identity of the fah12 cDNAas encoding a hydroxylase that hydroxylates both oleic acid to producericinoleic acid and also hydroxylates icosenoic acid to producelesquerolic acid. These results also provide additional evidence thatthe hydroxylase can be functionally expressed in a heterologous plantspecies in such a way that the enzyme is catalytically functional. Theseresults also demonstrate that expression of this hydroxylase-gene leadsto accumulation of ricinoleic, lesquerolic, densipolic and auricolicacids in a plant species that does not normally accumulate hydroxylatedfatty acids in extractable lipids.

The presence of lesquerolic acid in the transgenic plants wasanticipated in the previous patent application Ser. No. 08/320,982 basedon the biochemical evidence suggesting broad substrate specificity ofthe kappa hydroxylase. By contrast, the accumulation of densipolic andauricolic acids was less predictable. Since Arabidopsis does notnormally contain significant quantities of the non-hydroxylatedprecursors of these fatty acids which could serve as substrates for thehydroxylase, it appears that one or more of the three n-3 fatty aciddesaturases known in Arabidopsis (eg., fad3, fad7, fad8; reviewed inGibson et al., 1995) are capable of desaturating the hydroxylatedcompounds at the n-3 position. That is, densipolic acid is produced bythe action of an n-3 desaturase on ricinoleic acid. Auricolic acid isproduced by the action of an n-3 desaturase on lesquerolic acid. Becauseit is located in the endoplasmic reticulum, the fad3 desaturase isalmost certainly responsible. This can be tested in the future byproducing fah12-containing transgenic plants of the fad3-deficientmutant of Arabidopsis (similar experiments can be done with fad7 andfad8). It is also formally possible that the enzymes that normallyelongate 18:1^(cisΔ9) to 20:1^(cisΔ11) may elongate 12OH-18:1^(cisΔ9) to14OH-20:2^(cisΔ11), and 12OH-18:2^(cisΔ9,15) to 14OH-20:2^(cisΔ11,17).

The amount of the various fatty acids in seed, leaf and root lipids ofthe control and transgenic plants is also presented in Table 1. Althoughthe amount of hydroxylated fatty acids produced in this example is lessthan desired for commercial production of ricinoleate and otherhydroxylated fatty acids from plants, we envision numerous improvementsof this invention that will increase the level of accumulation ofhydroxylated fatty acids in plants that express the fah12 or relatedhydroxylase genes. Improvements in the level and tissue specificity ofexpression of the hydroxylase gene is envisioned. Methods to accomplishthis by the use of strong, seed-specific promoters such as the B. napusnapin promoter or the native promoters of the castor fah12 gene or thecorresponding hydroxylase gene from L. fendleri will be obvious to oneskilled in the art. Additional improvements are envisioned to involvemodification of the enzymes which cleave hydroxylated fatty acids fromphosphatidylcholine, reduction in the activities of enzymes whichdegrade hydroxylated fatty acids and replacement of acyltransferaseswhich transfer hydroxylated fatty acids to the sn-1, sn-2 and sn-3positions of glycerolipids. Although genes for these enzymes have notbeen described in the scientific literature, their utility in improvingthe level of production of hydroxylated fatty acids can be readilyenvisioned based on the results of biochemical investigations ofricinoleate synthesis.

Although Arabidopsis is not an economically important plant species, itis widely accepted by plant biologists as a model for higher plants.Therefore, the inclusion of this example is intended to demonstrate thegeneral utility of the invention described here and in the previousapplication Ser. No. 08/320,982 to the modification of oil compositionin higher plants. One advantage of studying the expression of this novelgene in Arabidopsis is the existence in this system of a large body ofknowledge on lipid metabolism, as well as the availability of acollection of mutants which can be used to provide useful information onthe biochemistry of fatty acid hydroxylation in plant species. Anotheradvantage is the ease of transposing any of the information obtained onmetabolism of ricinoleate in Arabidopsis to closely related species suchas the crop plants Brassica napus, Brassica juncea or Crambe abyssinicain order to mass produce ricinoleate, lesqueroleate or otherhydroxylated fatty acids for industrial use. The kappa hydroxylase isuseful for the production of ricinoleate or lesqueroleate in any plantspecies that accumulates significant levels of the precursors, oleicacid and icosenoic acid. Of particular interest are genetically modifiedvarieties that accumulate high levels of oleic acid. Such varieties arecurrently available for sunflower and Canola. Production of lesquerolicacid and related hydroxy fatty acids can be achieved in species thataccumulate high levels of icosenoic acid or other long chain monoenoicacids. Such plants may in the future be produced by genetic engineeringof plants that do not normally make such precursors. Thus, we envisionthat the use of the kappa hydroxylase is of general utility.

Example 2 Isolation of Lesquerella Kappa Hydroxylase Genomic CloneOverview

Regions of nucleotide sequence that were conserved in both the Castorkappa hydroxylase and the Arabidopsis fad2 Δ12 fatty acid desaturasewere used to design oligonucleotide primers. These were used withgenomic DNA from Lesquerella fendleri to amplify fragments of severalhomologous genes. These amplified fragments were then used ashybridization probes to identify full length genomic clones from agenomic library of L. fendleri.

Hydroxylated fatty acids are specific to the seed tissue of Lesquerellasp., and are not found to any appreciable extent in vegetative tissues.One of the two genes identified by this method was expressed in bothleaves and developing seeds and is therefore thought to correspond tothe Δ12 fatty acid desaturase. The other gene was expressed at highlevels in developing seeds but was not expressed or was expressed atvery low levels in leaves and is the kappa hydroxylase from thisspecies. The identity of the gene as a fatty acyl hydroxylase wasestablished by functional expression of the gene in yeast.

The identity of this gene will also be established by introducing thegene into transgenic Arabidopsis plants and showing that it causes theaccumulation of ricinoleic acid, lesquerolic acid, densipolic acid andauricolic acid in seed lipids. The promoter of this gene is also ofutility because it is able to direct expression of a gene specificallyin developing seeds at a time when storage lipids are accumulating. Thispromoter is, therefore, of great utility for many applications in thegenetic engineering of seeds, particularly in members of theBrassicacea.

The various steps involved in this process are described in detailbelow. Unless otherwise indicated, routine methods for manipulatingnucleic acids, bacteria and phage were as described by Sambrook et al.(1989).

Isolation of a Fragment of the Lesquerella Kappa Hydroxylase Gene

Oligonucleotide primers for the amplification of the L. fendleri kappahydroxylase were designed by choosing regions of high deduced amino acidsequence homology between the Castor kappa hydroxylase and theArabidopsis Δ12 desaturase (fad2). Because most amino acids are encodedby several different codons, these oligonucleotides were designed toencode all possible codons that could encode the corresponding aminoacids.

The sequence of these mixed oligonucleotides was:

Oligo 1: TAYWSNCAYMGNMGNVAYCA (SEQ ID NO:14) Oligo 2:RTGRTGNGCNACRTGNGTRTC (SEQ ID NO:15) (Where: Y = C + T; W = A + T; S = G+ C; N = A + G + C + T; M = A + C; R = A + G)

These oligonucleotides were used to amplify a fragment of DNA from L.fendleri genomic DNA by the polymerase chain reaction (PCR) using thefollowing conditions: Approximately 100 ng of genomic DNA was added to asolution containing 25 pmol of each primer, 1.5 U Taq polymerase(Boehringer Manheim), 200 uM of dNTPs, 50 mM KCl, 10 mM Tris.Cl (pH 9),0.1% (v/v) Triton X-100, 1.5 mM MgCl₂, 3% (v/v) formamide, to a finalvolume of 50 μl. Amplifications conditions were: 4 min denaturation stepat 94° C., followed by 30 cycles of 92° C. for 1 min, 55° C. for 1 min,72° C. for 2 min. A final extension step closed the program at 72° C.for 5 min.

PCR products of approximately 540 bp were observed followingelectrophoretic separation of the products of the PCR reaction inagarose gels. Two of these fragments were cloned into pBluescript(Stratagene) to give rise to plasmids pLesq2 and pLesq3. The sequence ofthe inserts in these two plasmids was determined by the chaintermination method. The sequence of the insert in pLesq2 is presented asFIG. 5 (SEQ ID NO:1) and the sequence of the insert in pLesq3 ispresented as FIG. 6 (SEQ ID NO:2). The high degree of sequence identitybetween the two clones indicated that they were both potentialcandidates to be either a Δ12 desaturase or a gamma hydroxylase.

Northern Analysis

In L. fendleri, hydroxylated fatty acids are found in large amounts inseed oils but are not found in appreciable amounts in leaves. Animportant criterion in discriminating between a fatty acyl desaturaseand kappa hydroxylase is that the kappa hydroxylase gene is expected tobe expressed more highly in tissues which have high level ofhydroxylated fatty acids than in other tissues. In contrast, all planttissues should contain mRNA for an ω6 fatty acyl desaturase sincediunsaturated fatty acids are found in the lipids of all tissues in mostor all plants.

Therefore, it was of great interest to determine whether the genecorresponding to pLesq2 was also expressed only in seeds, or is alsoexpressed in other tissues. This question was addressed by testing forhybridization of pLesq2 to RNA purified from developing seeds and fromleaves.

Total RNA was purified from developing seeds and young leaves of L.fendleri using an Rneasy RNA extraction kit (Qiagen), according to themanufacturer's instructions. RNA concentrations were quantified by UVspectrophotometry at λ=260 and 280 nm. In order to ensure even loadingof the gel to be used for Northern blotting, RNA concentrations werefurther adjusted after recording fluorescence under UV light of RNAsamples stained with ethidium bromide and run on a test denaturing gel.

Total RNA prepared as described above from leaves and developing seedswas electrophoresed through an agarose gel containing formaldehyde (Ibaet al., 1993). An equal quantity (10 μg) of RNA was loaded in bothlanes, and RNA standards (0.16-1.77 kb ladder, Gibco-BRL) were loaded ina third lane. Following electrophoresis, RNA was transferred from thegel to a nylon membrane (Hybond N+, Amersham) and fixed to the filter byexposure to UV light.

A ³²P-labelled probe was prepared from insert DNA of clone pLesq2 byrandom priming and hybridized to the membrane overnight at 52° C., afterit had been prehybridized for 2 h. The prehybridization solutioncontained 5×SSC, 10×Denhardt's solution, 0.1% SDS, 0.1M KPO₄ pH 6.8, 100μg/ml salmon sperm DNA. The hybridization solution had the same basiccomposition, but no SDS, and it contained 10% dextran sulfate and 30%formamide. The blot was washed once in 2×SSC, 0.5% SDS at 65° C. then in1×SSC at the same temperature.

Brief (30 min) exposure of the blot to X-ray film revealed that theprobe pLesq2 hybridized to a single band only in the seed RNA lane (FIG.7). The blot was re-probed with the insert from pLesq3 gene, which gavebands of similar intensity in the seed and leaf lanes (FIG. 7).

These results show that the gene corresponding to the clone pLesq2 ishighly and specifically expressed in seed of L. fendleri. In conjunctionwith knowledge of the nucleotide and deduced amino acid sequence, strongseed-specific expression of the gene corresponding to the insert inpLesq2 is a convincing indicator of the role of the enzyme in synthesisof hydroxylated fatty acids in the seed oil.

Characterization of a Genomic Clone of the Gamma Hydroxylase

Genomic DNA was prepared from young leaves of L. fendleri as describedby Murray and Thompson (1980). A Sau3AI-partial digest genomic libraryconstructed in the vector λDashII (Stratagene, 11011 North Torrey PinesRoad, La Jolla Calif. 92037) was prepared by partially digesting 500 μgof DNA, size-selecting the DNA on a sucrose gradient (Sambrook et al.,1989), and ligating the DNA (12 kb average size) to the BamHI-digestedarms of λDashII. The entire ligation was packaged according to themanufacturer's conditions and plated on E. coli strain XL1-Blue MRA-P2(Stratagene). This yielded 5×10⁵ primary recombinant clones. The librarywas then amplified according to the manufacturer's conditions. Afraction of the genomic library was plated on E. coli XL1-Blue andresulting plaques (150,000) were lifted to charged nylon membranes(Hybond N+, Amersham), according to the manufacturer's conditions. DNAwas crosslinked to the filters under UV in a Stratalinker (Stratagene).

Several clones carrying genomic sequences corresponding to the L.fendleri hydroxylase were isolated by probing the membranes with theinsert from pLesq2 that was PCR-amplified with internal primers andlabelled with ³²P by random priming. The filters were prehybridized for2 hours at 65° C. in 7% SDS, 1 mM EDTA, 0.25 M Na₂HPO₄ (pH 7.2), 1% BSAand hybridized to the probe for 16 hours in the same solution. Thefilters were sequentially washed at 65° C. in solutions containing2×SSC, 1×SSC, 0.5×SSC in addition to 0.1% SDS. A 2.6 kb Xba I fragmentcontaining the complete coding sequence for the gamma-hydroxylase andapproximately 1 kb of the 5′ upstream region was subcloned into thecorresponding site of pBluescript KS to produce plasmid pLesq-Hyd andthe sequence determined completely using an automatic sequencer by thedideoxy chain termination method. Sequence data was analyzed using theprogram DNASIS (Hitachi Company).

The sequence of the insert in clone pLesq-Hyd is shown in FIGS. 8A-B.The sequence entails 1855 bp of contiguous DNA sequence (SEQ ID NO:3).The clone encodes a 401 bp 5′ untranslated region (i.e., nucleotidespreceding the first ATG codon), an 1152 bp open reading frame, and a 302bp 3′ untranslated region. The open reading frame encodes a 384 aminoacid protein with a predicted molecular weight of 44,370 (SEQ ID NO:4).The amino terminus lacks features of a typical signal peptide (vonHeijne, 1985).

The exact translation-initiation methionine has not been experimentallydetermined, but on the basis of deduced amino acid sequence homology tothe Castor kappa hydroxylase (noted below) is thought to be themethionine encoded by the first ATG codon at nucleotide 402.

Comparison of the pLesq-Hyd deduced amino acid sequence with sequencesof membrane-bound desaturases and the castor hydroxylase (FIGS. 9A-B)indicates that pLesq-Hyd is homologous to these genes. This figure showsan alignment of the L. fendleri hydroxylase (SEQ ID NO:4) with thecastor hydroxylase (van de Loo et al. 1995), the Arabidopsis fad2 cDNAwhich encodes an endoplasmic reticulum-localized A12 desaturase (calledfad2) (Okuley et al., 1994), two soybean fad2 desaturase clones, aBrassica napus fad2 clone, a Zea mays fad2 clone and partial sequence ofa R. communis fad2 clone.

The high degree of sequence homology indicates that the gene productsare of similar function. For instance, the overall homology between theLesquerella hydroxylase and the Arabidopsis fad2 desaturase was 92.2%similarity and 84.8% identity and the two sequences differed in lengthby only one amino acid.

Southern Hybridization

Southern analysis was used to examine the copy number of the genes inthe L. fendleri genome corresponding to the clone pLesq-Hyd. Genomic DNA(5 μg) was digested with EcoR I, Hind III and Xba I and separated on a0.9% agarose gel. DNA was alkali-blotted to a charged nylon membrane(Hybond N+, Amersham), according to the manufacturer's protocol. Theblot was prehybridized for 2 hours at 65° C. in 7% SDS, 1 mM EDTA, 0.25M Na₂HPO₄ (pH 7.2), 1% BSA and hybridized to the probe for 16 hours inthe same solution with pLesq-Hyd insert PCR-amplified with internalprimers and labelled with ³²P by random priming. The filters weresequentially washed at 65° C. in solutions containing 2×SSC, 1×SSC,0.5×SSC in addition to 0.1% SDS, then exposed to X-ray film.

The probe hybridized with a single band in each digest of L. fendleriDNA (FIG. 10), indicating that the gene from which pLesq-Hyd wastranscribed is present in a single copy in the L. fendleri genome.

Expression of pLesq-Hyd in Transgenic Plants

There are a wide variety of plant promoter sequences which may be usedto cause tissue-specific expression of cloned genes in transgenicplants. For instance, the napin promoter and the acyl carrier proteinpromoters have previously been used in the modification of seed oilcomposition by expression of an antisense form of a desaturase (Knutsonet al. 1992). Similarly, the promoter for the β-subunit of soybeanβ-conglycinin has been shown to be highly active and to result intissue-specific expression in transgenic plants of species other thansoybean (Bray et al., 1987). Thus, although we describe the use of theL. fendleri kappa hydroxylase promoter in the examples described here,other promoters which lead to seed-specific expression may also beemployed for the production of modified seed oil composition. Suchmodifications of the invention described here will be obvious to oneskilled in the art.

Constructs for expression of L. fendleri kappa hydroxylase in plantcells are prepared as follows: A 13 kb SalI fragment containing thepLesq-Hyg gene was ligated into the XhoI site of binary Ti plasmidvector pSLJ44026 (Jones et al., 1992) (FIG. 11) to produce plasmidpTi-Hyd and transformed into Agrobacterium tumefaciens strains GV3101 byelectroporation. Strain GV3101 (Koncz and Schell, 1986) contains adisarmed Ti plasmid. Cells for electroporation were prepared as follows.GV3101 was grown in LB medium with reduced NaCl (5 g/l). A 250 mlculture was grown to OD₆₀₀=0.6, then centrifuged at 4000 rpm (SorvallGS-A rotor) for 15 min. The supernatant was aspirated immediately fromthe loose pellet, which was gently resuspended in 500 ml ice-cold water.The cells were centrifuged as before, resuspended in 30 ml ice-coldwater, transferred to a 30 ml tube and centrifuged at 5000 rpm (SorvallSS-34 rotor) for 5 min. This was repeated three times, resuspending thecells consecutively in 30 ml ice-cold water, 30 ml ice-cold 10%glycerol, and finally in 0.75 ml ice-cold 10% glycerol. These cells werealiquoted, frozen in liquid nitrogen, and stored at −80° C.Electroporations employed a Biorad Gene pulsar instrument using cold 2mm-gap cuvettes containing 40 μl cells and 1 μl of DNA in water, at avoltage of 2.5 KV, and 200 Ohms resistance. The electroporated cellswere diluted with 1 ml SOC medium (Sambrook et al., 1989, page A2) andincubated at 28° C. for 2-4 h before plating on medium containingkanamycin (50 mg/l).

Arabidopsis thaliana can be transformed with the Agrobacterium cellscontaining pTi-Hyd as described in Example 1 above. Similarly, thepresence of hydroxylated fatty acids in the transgeneic Arabidopsisplants can be demonstrated by the methods described in Example 1 above.

Constitutive Expression of the L. fendleri Hydroxylase in TransgenicPlants

A 1.5 kb EcoR I fragment from pLesq-Hyg comprising the entire codingregion of the hydroxylase was gel purified, then cloned into thecorresponding site of pBluescript KS (Stratagene). Plasmid DNA from anumber of recombinant clones was then restricted with Pst I, whichshould cut only once in the insert and once in the vector polylinkersequence. Release of a 920 bp fragment with Pst I indicated the rightorientation of the insert for further manipulations. DNA from one suchclone was further restricted with SalI, the 5′ overhangs filled-in withthe Klenow fragment of DNA polymerase I, then cut with Sac I. The insertfragment was gel purified, and cloned between the Sma I and Sac I sitesof pBI121 (Clontech) behind the Cauliflower Mosaic Virus 35S promoter.After checking that the sequence of the junction between insert andvector DNA was appropriate, plasmid DNA from a recombinant clone wasused to transform A. tumefaciens (GV3101). Kanamycin resistant colonieswere then used for in planta transformation of A. thaliana as previouslydescribed.

DNA was extracted from kanamycin resistant seedlings and used toPCR-amplify selected fragments from the hydroxylase using nestedprimers. When fragments of the expected size could be amplified,corresponding plants were grown in the greenhouse or on agar plates, andfatty acids extracted from fully expanded leaves, roots and dry seeds.GC-MS analysis was then performed as previously described tocharacterize the different fatty acid species and detect accumulation ofhydroxy fatty acids in transgenic tissues.

Expression of the Lesquerella Hydroxylase in Yeast

In order to demonstrate that the cloned L. fendleri gene encoded anoleate-12 hydroxylase, the gene was expressed in yeast cells undertranscriptional control of an inducible promoter and the yeast cellswere examined for the presence of hydroxylated fatty acids by GC-MS.

In a first step, a lambda genomic clone containing the L. fendlerihydroxylase gene was cut with EcoRI, and a resulting 1400 bp fragmentcontaining the coding sequence of the hydroxylase gene was subcloned inthe EcoRI site of the pBluescript KS vector (Stratagene). This subclone,pLesqcod, contains the coding region of the Lesquerella hydroxylase plussome additional 3′ sequence.

In a second step, pLesqcod was cut with HindIII and XbaI, and the insertfragment was cloned into the corresponding sites of the yeast expressionvector pYes2 (In Vitrogen; FIG. 12). This subclone, pLesqYes, containsthe L. fendleri hydroxylase in the sense orientation relative to the 3′side of the Gal1 promoter. This promoter is inducible by the addition ofgalactose to the growth medium, and is repressed upon addition ofglucose. In addition, the vector carries origins of replication allowingthe propagation of pLesqYes in both yeast and E. coli.

Transformation of S. cerevisiae Host Strain CGY2557

Yeast strain CGY2557 (MATα, GAL⁺, ura3-52, leu2-3, trp1, ade2-1, lys2-1,hiss, can1-100) was grown overnight at 28° C. in YPD liquid medium (10 gyeast extract, 20 g bacto-peptone, 20 g dextrose per liter), and analiquot of the culture was inoculated into 100 ml fresh YPD medium andgrown until the OD₆₀₀ of the culture was 1. Cells were then collected bycentrifugation and resuspended in about 200 μl of supernatant. 40 μlaliquots of the cell suspension were then mixed with 1-2 μg DNA andelectroporated in 2 mm-gap cuvettes using a Biorad Gene Pulserinstrument set at 600 V, 200Ω, 25 μF. 160 μl YPD was added and the cellswere plated on selective medium containing glucose. Selective mediumconsisted of 6.7 g yeast nitrogen base (Difco), 0.4 g casamino acids(Difco), 0.02 g adenine sulfate, 0.03 g L-leucine, 0.02 g L-tryptophan,0.03 g L-lysine-HCl, 0.03 g L-histidine-HCl, 2% glucose, water to 1liter. Plates were solidified using 1.5% Difco Bacto-agar. Transformantcolonies appeared after 3 to 4 days incubation at 28° C.

Expression of the L. fendleri Hydroxylase in Yeast

Independent transformant colonies from the previous experiment were usedto inoculate 5 ml of selective medium containing either 2% glucose (generepressed) or 2% galactose (gene induced) as the sole carbon source.Independent colonies of CGY2557 transformed with pYES2 containing noinsert were used as controls.

After 2 days of growth at 28° C., an aliquot of the cultures was used toinoculate 5 ml of fresh selective medium. The new culture was placed at16° C. and grown for 9 days.

Fatty Acid Analysis of Yeast Expressing the L. fendleri Hydroxylase

Cells from 2.5 ml of culture were pelleted at 1800 g, and thesupernatant was aspirated as completely as possible. Pellets were thendispersed in 1 ml of 1 N methanolic HCl (Supelco, Bellafonte, Pa.).Transmethylation and derivatization of hydroxy fatty acids wereperformed as described above. After drying under nitrogen, samples wereredissolved in 50 μL chloroform before being analyzed by GC-MS. Sampleswere injected into an SP2330 fused-silica capillary column (30 m×0.25 mmID, 0.25 μm film thickness, Supelco). The temperature profile was100-160° C., 25° C./min, 160-230° C., 10° C./min, 230° C., 3 min,230-100° C., 25° C./min. Flow rate was 0.9 ml/min. Fatty acids wereanalyzed using a Hewlett-Packard 5971 series Msdetector.

Gas chromatograms of derivatized fatty acid methyl esters from inducedcultures of yeast containing pLesqYes contained a novel peak that elutedat 7.6 min (FIG. 13). O-TMS methyl ricinoleate eluted at exactly thesame position on control chromatograms. This peak was not present incultures lacking pLesqYes or in cultures containing pLesqYes grown onglucose (repressing conditions) rather than galactose (inducingconditions). Mass spectrometry of the peak (FIG. 13) revealed that thepeak has the same spectrum as O-TMS methyl ricinoleate. Thus, on thebasis of chromatographic retention time and mass spectrum, it wasconcluded that the peak corresponded to O-TMS methyl ricinoleate. Thepresence of ricinoleate in the transgenic yeast cultures confirms theidentity of the gene as a kappa hydroxylase of this invention.

Example 3 Obtaining Other Plant Fatty Acyl Hydroxylases

In a previous patent application, we described the ways in which thecastor fah12 sequence could be used to identify other kappa hydroxylasesby methods such as PCR and heterologous hybridization. However, becauseof the high degree of sequence similarity between Δ12 desaturases andkappa hydroxylases, prior art does not teach how to distinguish betweenthe two kinds of enzymes without a functional test such as demonstratingactivity in transgenic plants or another suitable host (e.g., transgenicmicrobial or animal cells). The identification of the L. fendlerihydroxylase provided for the development of criteria by which ahydroxylase and a desaturase may be distinguished solely on the basis ofdeduced amino acid sequence information.

FIGS. 9A-B show a sequence alignment of the castor and L. fendlerihydroxylase sequences with the castor hydroxylase sequence and allpublicly available sequences for all plant microsomal Δ12 fatty aciddesaturases. Of the 384 amino acid residues in the castor hydroxylasesequence, more than 95% are identical to the corresponding residue in atleast one of the desaturase sequences. Therefore, none of these residuesare responsible for the catalytic differences between the hydroxylaseand the desaturases. Of the remaining 16 residues in the castorhydroxylase and 14 residues in the Lesquerella hydroxylase, all but sixrepresent instances where the hydroxylase sequence has a conservativesubstitution compared with one or more of the desaturase sequences, orthere is wide variability in the amino acid at that position in thevarious desaturases. By conservative, we mean that the following aminoacids are functionally equivalent: Ser/Thr, Ile/Leu/Val/Met, Asp/Glu.Thus, these structural differences also cannot account for the catalyticdifferences between the desaturases and hydroxylases. This leaves justsix amino acid residues where both the castor hydroxylase and theLesquerella hydroxylase differ from all of the known desaturases andwhere all of the known microsomal Δ12 desaturases have the identicalamino acid residue. These residues occur at positions 69, 111, 155, 226,304 and 331 of the alignment in FIG. 9. Therefore, these six sitesdistinguish hydroxylases from desaturases. Based on this analysis, weclaim that any enzyme with greater than 60% sequence identity to one ofthe enzymes listed in FIG. 9 can be classified as a hydroxylase if itdiffers from the sequence of the desaturases at these six positions.Because of slight differences in the number of residues in a particularprotein, the numbering may vary from protein to protein but the intentof the number system will be evident if the protein in question isaligned with the castor hydroxylase using the numbering system shownherein. Thus, in conjunction with the methods for using the Lesquerellahydroxylase gene to isolate homologous genes, the structural criteriondisclosed here teaches how to isolate and identify plant kappahydroxylase genes for the purpose of genetically modifying fatty acidcomposition as disclosed herein and in the previous application Ser. No.08/320,982.

In considering which of the six substitutions are solely or primarilyresponsible for the difference in catalytic activity of the hydroxylasesof this invention and the desaturases, we consider it likely that thesubstitution of a Phe for a Tyr at position 226 may be solelyresponsible for this difference in catalytic activity because of theknown participation of tyrosine radicals in enzyme catalysis. Othersubstitutions, such as the Ala for Ser at position 331 may have effectsat modulating the overall rate of the reaction. On this basis weenvision creating novel kappa hydroxylases by site directed mutagenesisof Δ12 desaturases. We also envision converting Δ15 desaturases and Δ9desaturases to hydroxylases by similar use of site-directed mutagenesis.

Example 4 Using Hydroxylases to Alter the Level of Fatty AcidUnsaturation

Evidence that kappa hydroxylases of this invention can be used to alterthe level of fatty acid unsaturation was obtained from the analysis oftransgenic plants that expressed the castor hydroxylase under control ofthe Cauliflower mosaic virus promoter. The construction of the plasmidsand the production of transgenic Arabidopsis plants was described inExample 1 (above). The fatty acid composition of seed lipids from wildtype and six transgenic lines (1-2/a, 1-2/b, 1-3/b, 4F, 7E, 7F) is shownin Table 2.

TABLE 2 Fatty acid composition of lipids from Arabidopsis seeds. Fattyacid WT 1-2/a 1-2/b 1-3/b 4F 7E 7F 16:0 10.3 8.6 9.5 8.4 8.1 8.4 9 18:03.5 3.8 3.9 3.3 3.5 3.8 4.2 18:1 14.7 33 34.5 25.5 27.5 30.5 28.5 18:232.4 16.9 21 27.5 21.1 20.1 19.8 18:3 13.8 — 14.4 14.8 — — — 20:0 1.31.6 1 1.1 2.4 1.8 2 20:1 22.5 — 14.1 17.5 — — — 18:3 — 31.2 — — 32.130.8 30.6 20:1 * Ricinoleic 0 0.6 0 0.1 0.2 0.7 0.9 Densipolic 0 0.6 00.1 0.2 0.5 0.6 Lesquerolic 0 0.2 0 0 0.2 0.2 0.6 Auricolic 0 0.1 0 0 00.1 0.1 The asterisk (*) indicates that for some of these samples, the18:3 and 20:1 peaks overlapped on the gas chromatograph and, therefore,the total amount of these two fatty acids is reported.

The results in Table 2 show that expression of the castor hydroxylase intransgenic Arabidopsis plants caused a substantial increase in theamount of oleic acid (18:1) in the seed lipids and an approximatelycorresponding decrease in the amount of linoleic acid (18:2). Theaverage amount of oleic acid in the six transgenic lines was 29.9%versus 14.7% in the wild type.

The mechanism by which expression of the castor hydroxylase gene causesincreased accumulation of oleic acid is not known. An understanding ofthe mechanism is not required in order to exploit this invention for thedirected alteration of plant lipid fatty acid composition. Furthermore,it will be recognized by one skilled in the art that many improvementsof this invention may be envisioned. Of particular interest will be theuse of other promoters which have high levels of seed-specificexpression.

Since hydroxylated fatty acids were not detected in the seed lipids oftransgenic line 1-2b, it seems likely that it is not the presence ofhydroxylated fatty acids per se that causes the effect of the castorhydroxylase gene on desaturase activity. We speculate that there may bea protein-protein interaction between the hydroxylase and the Δ12-oleatedesaturase or another protein required for the overall reaction (eg.,cytochrome b5) or for the regulation of desaturase activity. We envisionthat the interaction between the hydroxylase and this other proteinsuppresses the activity of the desaturase. For instance, the quaternarystructure of the membrane-bound desaturases has not been established. Itis possible that these enzymes are active as dimers or as multimericcomplexes containing more than two subunits. Thus, if dimers ormultimers formed between the desaturase and the hydroxylase, thepresence of the hydroxylase in the complex may disrupt the activity ofthe desaturase. This general hypothesis will be tested directly by theproduction of transgenic plants in which the hydroxylase enzyme has beenrendered inactive by the elimination of one or more of the histidineresidues that have been proposed to bind iron molecules required forcatalysis. Several of these histidine residues have been shown to beessential for catalysis by site directed mutagenesis (Shanklin et al.,1994). Codons encoding histidine residues in the castor hydroxylase genedescribed in U.S. patent application Ser. No. 08/320,982 will be changedto alanine residues as described by Shanklin et al. (1994). The modifiedgenes will be introduced into transgenic plants of Arabidopsis andpossibly other species such as tobacco by the methods described inExample 1 of this application or in Example 1 of the original version ofthis application (U.S. application Ser. No. 08/320,982).

In order to examine the effect on all tissues, the strong constitutivecauliflower mosaic virus promoter will be used to cause transcription ofthe modified genes. However, it will be recognized that in order tospecifically examine the effect of expression of the mutant gene on seedlipids, a seed-specific promoter such as the B. napus napin promoter orthe promoter described in Example 2, above, may be used. An expectedoutcome is that expression of the inactive hydroxylase protein intransgenic plants will inhibit the activity of the endoplasmicreticulum-localized Δ12-desaturase. Maximum inhibition will be obtainedby expressing high levels of the mutant protein.

In a further embodiment of this invention, we envision that mutationsthat inactivate other hydroxylases, such as the Lesquerella hydroxylaseof this invention, will also be useful for decreasing the amount ofendoplasmic reticulum-localized Δ12-desaturase activity in the same wayas the castor gene. In a further embodiment of this invention, we alsoenvision that similar mutations of desaturase genes may be used toinactivate endogenous desaturases. Thus, we envision that expression ofcatalytically inactive fad2 gene from Arabidopsis in transgenicArabidopsis will inhibit the activity of the endogenous fad2 geneproduct.

Similarly, we envision that expression of the catalytically inactiveforms of the Δ12-desaturase from Arabidopsis or other plants intransgenic soybean in transgenic rapeseed, Crambe, Brassica juncea,Canola, flax, sunflower, safflower, cotton, cuphea, soybean, peanut,coconut, oil palm and corn will lead to inactivation of endogenousΔ12-desaturase activity in these species. In a further embodiment ofthis invention we envision that expression of catalytically inactiveforms of other desaturases such as the Δ15-desaturases will lead toinactivation of the corresponding desaturases.

Whatever the precise basis for the inhibitory effect of the castorhydroxylase on desaturation, because the castor hydroxylase has very lownucleotide sequence homology (i.e., about 67%) to the Arabidopsis fad2gene (encoding the endoplasmic reticulum-localized Δ12-desaturase) weenvision that the inhibitory effect of this gene, which we provisionallycall “protein-mediated inhibition” (“protibition”), will have broadutility because it does not depend on a high degree of nucleotidesequence homology between the transgene and the endogenous target gene.In particular, we envision that the castor hydroxylase may be used toinhibit the endoplasmic reticulum-localized Δ12-desaturase activity ofall higher plants. Of particular relevance are those species used foroil production. These include but are not limited to rapeseed, Crambe,Brassica juncea, Canola, flax, sunflower, safflower, cotton, cuphea,soybean, peanut, coconut, oil palm and corn.

CONCLUDING REMARKS

By the above examples, demonstration of critical factors in theproduction of novel hydroxylated fatty acids by expression of a kappahydroxylase gene from Castor in transgenic plants is described. Inaddition, a complete cDNA sequence of the Lesquerella fendleri kappahydroxylase is also provided. A full sequence of the castor hydroxylaseis also given with various constructs for use in host cells. Throughthis invention, one can obtain the amino acid and nucleic acid sequenceswhich encode plant fatty acyl hydroxylases from a variety of sources andfor a variety of applications. Also revealed is a novel method by whichthe level of fatty acid desaturation can be altered in a directed waythrough the use of genetically altered hydroxylase or desaturase genes.

All publications mentioned in this specification are indicative of thelevel of skill of those skilled in the art to which this inventionpertains. All publications are herein incorporated by reference to thesame extent as if each individual publication wasp specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain is changes andmodifications may be practiced within the scope of the appended claims.

REFERENCES

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1-35. (canceled)
 36. A seed of a Brassicaceae plant, wherein oil of theseed comprises ricinoleic acid.
 37. A plant comprising the seed of claim36.
 38. The plant of claim 36, wherein the plant is selected from thegroup consisting of Brassica napus, Brassica juncea, Brassica nigra andCrambe abyssinica.
 39. The plant of claim 37, wherein the plant isselected from the group consisting of Brassica napus, Brassica juncea,Brassica nigra and Crambe abyssinica.
 40. A seed oil obtained from theseed of claim 36, wherein the seed oil comprises ricinoleic acid.
 41. Aseed oil obtained from seeds of a Brassicaceae plant, wherein the seedoil comprises ricinoleic acid.
 42. An oil comprising a seed oil from aBrassicaceae plant, wherein the seed oil comprises ricinoleic acid.